Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila.